This web page was produced as an
assignment for an undergraduate course at Davidson College.

General
Information

Ligases are
enzymes that seal breaks in the phosphate-sugar backbone of DNA and RNA. There
are two main classes of DNA ligases, those that use NAD+ as a cofactor (only in
bacteria), and those that use ATP as a cofactor (eukaryotes and viruses) (Kahn,
2003). Mammalian cells have four types of DNA ligase, which together accomplish
three main functions: joining Okazaki fragments, sealing repairs, and sealing
recombination fragments (Purves et al., 2001).

DNA ligase I: connects Okazaki fragments of the lagging strand in
DNA replication, and can also seal some repair and recombination fragments (Wei
et al., 1995).

DNA ligase II: an alternatively spliced form of DNA ligase III that
is only expressed in non-dividing cells.

DNA ligase III: works with protein XRCC1, which is a DNA repair
protein. DNA ligase III is a primary agent in sealing base excision-repairs and
recombination fragments.

DNA ligase IV: works with protein XRCC4, which is another DNA repair
protein. DNA ligase IV is also important in sealing base excision-repairs
and recombination fragments, especially during development (Schär et al.,
1997).

Mechanism

The following diagrams and
descriptions are of the three main steps of ligation among all types of DNA
ligases. Notice how ligase follows the general properties of enzymes, solely
providing a docking area for the reaction between the AMP and DNA.

1. Adenylation of DNA
ligase:

The side chain of lysine 34 in
ligase forms a bond with ATP, where ATP kicks off two phosphate groups to
become an AMP-ligase complex (Kahn, 2003).

Figure 1. Adenylation of DNA
ligase by ATP.

2. Activation of 5’
phosphate:

The monophosphate of the
AMP-ligase complex forms a bond with the 5’ phosphate of the broken strand.
This bonding activates the 5’ phosphate group for the next step.

Figure 2. Activation of 5’ phosphate
mediated by AMP-ligase complex.

3. Displacement of AMP
connects the broken strand:

Now that the 5’ phosphate has
been activated by the AMP-ligase complex, the 3’ hydroxyl group attacks the 5’
phosphate and forms a new bond, releasing the AMP. Ligase is crucial for
holding the complex together in the necessary orientation.

Figure 4. Overall reaction of the ligation
of a broken DNA strand to form a new phosphodiester bond between the adjacent
phosphate and sugar.

Using Ligation
as a Molecular Tool

Figure 5. Chime image of ATP-dependent DNA ligase from
bacteriophage T7 complex with ATP. Notice the location of lysine 34 within
ligase that will eventually form a covalent bond with AMP. Click
Here for Source: PDB

T7 DNA ligase is approximately half the size of human DNA ligase, having a
molecular weight of 41 KdA (Kahn, 2003). However, T7 DNA ligase is a versatile
tool for molecular biologists because it shares common structure with a wide
spectrum of ligases from different species. As mentioned before, ligases seal
breaks in the phosphate-sugar backbone of adjacent nucleotides in DNA or RNA.
In creating expression
vectors, and many synthetic DNA fragments, ligases are essential in linking
the pieces of the puzzle. The most efficient way for a ligase to link DNA
fragments is to connect two sticky ends together from a restriction digest. The
hydrogen bonds between the complementary bases aid ligase in holding the two
ends together while the phosphodiester bond is formed. Although ligases are
capable of connecting two blunt ended DNA fragments together, the reaction is
much slower and prone to greater error. Blunt end ligation should have controls
to demonstrate that the proper fragments were ligased because the specificity
decreases from sticky end ligation. Ligations are relatively fast reactions at
16o C, usually requiring 5-30 minutes to completely ligate a sample
(Kahn, 2003). One limitation to ligation is that it requires a 5’ phosphate on
the DNA fragment to work at all, however T4 polynucleotide kinase and ATP can
usually phosphorylate the 5’ carbon in the broken DNA strand (Kahn, 2003).